1) Valutazione caratteristiche neurochimiche e quantificazione dei neuroni del SNE dell’ileo di cani ctrl e cani affetti da infiammazione intestinale (spontanea). Prelevati campioni di ileo da 5 cani ctrl e 8 cani patol ed ottenute criosezioni sottoposte ad immunoistochimica. - nNOS-IR MP ctrl 33±15% (178/639 cell, n=5) vs patol 24±5% (528/2031 cell, n=7) (P=0,156). nNOS-IR SMP ctrl 8±5% (40/527 cell, n=5) vs patol 7±2% (69/1007 cell, n=4) (P= 0,735). - VIP-IR MP ctrl 6±4% (69/993 cell) vs patol 16±9% (281/1958 cell, n=8) (P=0,027*). VIP-IR SMP ctrl 29±8% (300/993 cell) vs patol 30±13% (522/1630 cell, n=7) (P=0,891,) - SP-IR MP ctrl 15±8% (209/1332 cell, n=5) vs patol 17±9% (437/2053 cell, n=8) (P=0,741). SP-IR SMP ctrl 26±7% (464/1598 cell, n=5) vs patol 24±13% (592/2024 cell, n=6) (P=0,752). - CGRP-IR MP ctrl 8±9% (41/543 cell, n=5) vs patol 16±11% (259/1444 cell, n=8) (P=0,152). CGRP-IR SMP ctrl 7±8% (32/754 cell, n=5) vs patol 14±12% (194/1138 cell, n=7) (P=0,230) - Calb-IR MP ctrl 14±9% (76/580 cell, n=5) vs patol 16±7% (324/2055 cell, n=8)(P=0,596). CGRP-IR SMP ctrl di 22±7% (218/975 cell, n=5) vs patol 23±7% (261/1207 cell, n=7) (P=0,767). Questo rappresenta il primo studio esistente sulle caratteristiche neurochimiche delle sottopopolazioni di neuroni in cani ctrl e cani affetti da patol gastroenterica. 2) Valutazione degli effetti del diabete mellito di tipo I sul SNE dell’antro pilorico e dell’ileo di cane. Prelevati campioni di antro e ileo di 8 cani ctrl e 5 patol ed ottenute criosezioni sottoposte ad immunoistochimica. nNOS-IR MP ctrl antro 30±6%, (902/3129 cellule), ileo 29±5% (795/2800 cellule). nNOS-IR MP DM antro 25±2% (727/2926 cellule), ileo (19±5%; 308/1508 cellule).(P antro=0.112), (P ileo=0.006). Questi risultati indicano che il DM determini un’alterazione dell’innervazione nitrergica maggiore rispetto nell’ileo rispetto allo stomaco. / 1) Study of effects of spontaneous inflammatory bowel disease in SNE of dogs. Specimens of ileum were collected from 5 control and 8 affected dogs, use for immunohistochemistry on the cryosections - nNOS-IR MP ctrl 33±15% (178/639 cell, n=5) vs patol 24±5% (528/2031 cell, n=7) (P=0,156). nNOS-IR SMP ctrl 8±5% (40/527 cell, n=5) vs patol 7±2% (69/1007 cell, n=4) (P= 0,735). - VIP-IR MP ctrl 6±4% (69/993 cell) vs patol 16±9% (281/1958 cell, n=8) (P=0,027*). VIP-IR SMP ctrl 29±8% (300/993 cell) vs patol 30±13% (522/1630 cell, n=7) (P=0,891,) - SP-IR MP ctrl 15±8% (209/1332 cell, n=5) vs patol 17±9% (437/2053 cell, n=8) (P=0,741). SP-IR SMP ctrl 26±7% (464/1598 cell, n=5) vs patol 24±13% (592/2024 cell, n=6) (P=0,752). - CGRP-IR MP ctrl 8±9% (41/543 cell, n=5) vs patol 16±11% (259/1444 cell, n=8) (P=0,152). CGRP-IR SMP ctrl 7±8% (32/754 cell, n=5) vs patol 14±12% (194/1138 cell, n=7) (P=0,230) - Calb-IR MP ctrl 14±9% (76/580 cell, n=5) vs patol 16±7% (324/2055 cell, n=8)(P=0,596). CGRP-IR SMP ctrl di 22±7% (218/975 cell, n=5) vs patol 23±7% (261/1207 cell, n=7) (P=0,767). This is the first study about neurochemical characteristics of neuron subsets in control dogs and in gastrointestinal disease afflicted dogs. 2) Study the effects of spontaneous DM on the nitrergic neurons of the MP of the canine gastric antrum and ileum. Specimens of gastric antrum and ileum from eight control dogs and five insulin-dependent DM dogs were collected. MP neurons were immunohistochemically identified with the anti-HuC/HuD and nNOS antibody. nNOS-IR MP stomachs control dogs was 30±6%, in the DM was 25±2% (P=0.112). nNOS-IR MP ileum control dogs was 29±5%, in the DM was significantly reduced 19±5% (P=0.006). These findings indicate that DM in dogs alters intestinal nitrergic innervation more rather than the gastric one.

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L’infezione da virus dell’ epatite E (HEV) nei suini e nell’uomo è stata segnalata in diversi Paesi. Nei suini, il virus causa infezioni asintomatiche, mentre nell’uomo è responsabile di epidemie di epatite ad andamento acuto nei Paesi a clima tropicale o subtropicale con condizioni igieniche scadenti, di casi sporadici in quelli sviluppati. HEV è stato isolato anche in diversi animali e l’analisi nucleotidica degli isolati virali di origine animale ha mostrato un elevato grado di omologia con i ceppi di HEV umani isolati nelle stesse aree geografiche, avvalorando l’ipotesi che l'infezione da HEV sia una zoonosi. In America del Sud HEV suino è stato isolato per la prima volta in suini argentini nel 2006, mentre solo dal 1998 esistono dati sull’ infezione da HEV nell’uomo in Bolivia. In questa indagine è stato eseguito uno studio di sieroprevalenza in due comunità rurali boliviane e i risultati sono stati confrontati con quelli dello studio di sieroprevalenza sopra menzionato condotto in altre zone rurali della Bolivia. Inoltre, mediante Nested RT-PCR, è stata verificata la presenza di HEV nella popolazione umana e suina. La sieroprevalenza per anticorpi IgG anti-HEV è risultata pari al 6,2%, molto simile a quella evidenziata nello studio precedente. La prevalenza maggiore (24%) si è osservata nei soggetti di età compresa tra 41 e 50 anni, confermando che l’ infezione da HEV è maggiore fra i giovani-adulti. La ricerca di anticorpi anti HEV di classe IgM eseguita su 52 sieri ha fornito 4 risultati positivi. Il genoma virale è stato identificato in uno dei 22 pool di feci umane e l'esame virologico di 30 campioni individuali fecali e 7 individuali di siero ha fornito rispettivamente risultati positivi in 4/30 e 1/7. La Nested RT-PCR eseguita sui 22 pool di feci suine ha dato esito positivo in 7 pool. L’analisi delle sequenze genomiche di tutti gli amplificati ha consentito di stabilire che gli isolati umani appartenevano allo stesso genotipo III di quelli suini e presentavano con questi una elevata omologia aminoacidica (92%). / Hepatitis E Virus (HEV) infection of swine and human has been reported in various countries. In humans the virus causes hepatitis E epidemics in areas with tropical or subtropical climate and poor sanitary conditions, and sporadic cases in developed countries. HEV infection in swine is quite spread and asymptomatic. HEV infections have been reported in different animals and animal strains were found to be closely related to human HEV strains from the same area, suggesting that hepatitis E may be a zoonosis. In South America, HEV of swine origin has been isolated for the first time in 2006 in Argentina, while human infection in Bolivia has been reported only in 1998 based on a serological investigation carried on in rural communities. In this study we have investigated the presence of HEV infection in two rural Bolivian communities and the results were compared with those of the aforementioned study. Anti-HEV IgG antibodies were detected in 6,2% of the sera examined. The highest seroprevalence (24%) was detected in the individuals aged from 41 to 50 years, confirming that the infection is more prevalent in young-adult individuals. Anti-IgM antibodies were detecte in 4 serum individuals out of 52 examined. HEV has been detected by means of Nested RT-PCR in 7 out of 22 pools of swine feces examined, in 1 pool out of 22 of human feces, 7 individual samples of human feces out of 30 and in 1 sample of human serum out of 7. Phylogenetic analysis demonstrated that human and swine HEV isolates could be included all into the genotype III and shared high degree of homology.

Parapoxvirus (PPV) are member of a genus in the family poxviridae which currently encompasses four species: the prototype orf virus (OV), bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV) and parapoxvirus of New Zealand red deer (PVNZ). PPVs cause widespread, but localized diseases of small and large ruminants and they can also be transmitted to man. Knowledge of the molecular biology of PPV is still limited as compared to orthopoxviruses, especially vaccinia virus (VACV). The PPV genome displays a high G+C content and relatively small size for poxvirus. Coventional electron microscopy displays PPV virions with ovoid shape and slightly smaller in size than the brickshaped orthopoxviruses. The most striking feature, which readily enables identification of PPV, is a tubule-like structure that surrounds the particle in a spiral fashion. PPV genome organization and content is very similar to that of other poxviruses, the central region contain 88 genes which are present in all poxviruse, in contrast the terminal regions are variable and contain a set of genes unique to the genus PPV. Genes in the near-terminal regions of the genome are frequently not essential for growth in cultured cells encoding factors with important roles in virushost interactions including modulating host immune responses and determining host range. Recently it was suggested that the open reading frames (ORFs) 109 and 110 of the OV genome have a major role in determining species specificity during natural infection in sheep and goats. This hypothesis is based on the analysis of a few number of sequences of different sheep and goats viral isolates. PPV replicate into the cytoplasm of infected cells and produce three structurally different infectious particles: the intracellular mature virions (IMV), intracellular enveloped virions (IEV) and the extracellular enveloped virions (EEV). The vaccinia A33R and A34R hotologue proteins encoded by the ORFS 109 and 110 are expressed in the envelope of the IEV and EEV. The F1L immunodominant protein of orf virus is the major component of the surface tubule structure of the IMV and can post-translationaly insert into membranes via Cterminal, hydrofobic anchor sequence like its orthologue VACV H3L protein. Moreover the F1L protein binds to glycosaminoglycans on the cell surface and has an important role in IMV adsorption to mammalian cells. In this study we investigated the morphogenesis of the PPV through the construction of a mutant virus deleted of the F1L protein. A study of the deleted virus life cycle was conducted in different type of cells and its morphology was observed with electron microscopy. It was demonstared that F1L protein have important role in morphogenesis and infectivity. Moreover it is essential to determine the spiral fashion of the tubule like structure of the virion surface. Some pathogenetic aspects of the PPV infection were studied, in particular the protein implicated in the host range were analysed in detail. An experimental infection with OV and PCPV was conducted in goats and sheep. After infection, the severity of the lesions were comparable in both the animal species. The OV did not result in severe disease neither in sheep nor in goats, suggesting that host factors, rather than virus strain characteristics, may play an important role in the pathogenesis of the Parapoxvirus infections. The PCPV failed to produce any lesion in both sheep and goats, ruling out the possibility of any recombination between PCPV and OV during natural infection in these animal species. The phylogenetic analysis of the ORFs 109 and 110 from several goats and sheep viral isolates showed a clustering based on the antigenic content of the protein that was independent from species and geographic origin.

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From September 2005 to December 2006, in order to define the prevalence of Helicobacter pullorum in broiler chickens, laying hens and turkey, a total of 365 caecum contents of animals reared in 76 different farms were collected at the slaughterhouse. A caecum content of a ostrich was also sampled. In addition, with the aim of investigating the occurrence of H. pullorum in humans, 151 faeces were collected at the Sant’Orsola-Malpighi University Hospital of Bologna from patients suffering of gastroenteritis. A modified Steele–McDermott membrane filter method was used. Gram-negative curved rod bacteria were preliminary identified as H. pullorum by a PCR assay based on 16S rRNA, then subjected to a RFLP-PCR assay to distinguish between H. pullorum and H. canadensis. One isolate from each farm was randomly selected for phenotypic characterization by biochemical methods and 1D SDSPAGE analysis of whole cell proteins profiles. Minimum Inhibitory Concentration (MIC) for seven different antibiotics were also determined by agar dilution method. Moreover, to examine the intraspecific genomic variability, two strains isolated from 17 different farms were submitted to genotyping by Pulse-Field Gel Electrophoresis (PFGE). In order to assess the molecular basis of fluorquinolone resistance in H. pullorum, gyrA of H. pullorum CIP 104787T was sequenced and nucleotide sequences of the Quinolone Resistance Determining Region (QRDR) of a total of 18 poultry isolates, with different MIC values for ciprofloxacin and nalidixic acid, were compared. According to the PCR and PCR-RFLP results, 306 out of 366 animals examined were positive for H. pullorum (83,6%) and 96,1% of farms resulted infected. All positive samples showed a high number of colonies (>50) phenotipically consistent with H. pullorum on the first isolation media, which suggests that this microrganism, when present, colonizes the poultry caecum at an elevate load. No human sample resulted positive for H. pullorum. The 1D SDS-PAGE whole protein profile analysis showed high similarity among the 74 isolates tested and with the type strain H. pullorum CIP 104787T. Regarding the MIC values, a monomodal distribution was found for ampicillin, chloramphenicol, gentamicin and nalidixic acid, whereas a bimodal trend was noticed for erythromycin, ciprofloxacin and tetracycline (indicating an acquired resistance for these antibiotics). Applying the breakpoints indicated by the CSLI, we may assume that all the H. pullorum tested are sensitive only to gentamicin. The intraspecific genomic variability observed in this study confirm that this species don’t have a clonal population structure, as motioned by other autors. The 2490 bp gyrA gene of H. pullorum CIP104787T with an Open Reading Frame (ORF) encoding a polypeptide of 829 amino acids was for the first time sequenced and characterized. All ciprofloxacin resistant poultry isolates showed ACA®ATA (Thr®Ile) substitution at codon 84 of gyrA corresponding to codons of gyrA 86, 87 and 83 of the Campylobacter jejuni, H. pylori and Escherichia coli, respectively. This substitution was functionally confirmed to be associated with the ciprofloxacin resistant phenotype of poultry isolates. This is the first report of isolation of H. pullorum in turkey and in ostrich, indicating that poultry species are the reservoir of this potential zoonotic microorganisms. In order to understand the potential role as food-borne human pathogen of H. pullorum, further studies must be carried on.